Unique cistrome defined as CsMBE is strictly required for Nrf2-sMaf heterodimer function in cytoprotection

Nrf2-small Maf (sMaf) heterodimer is essential for the inducible expression of cytoprotective genes upon exposure to oxidative and xenobiotic stresses. While the Nrf2-sMaf heterodimer recognizes DNA sequences referred to as the antioxidant/electrophile responsive element (ARE/EpRE), we here define these DNA sequences collectively as CNC-sMaf binding element (CsMBE). In contrast, large and small Maf proteins are able to form homodimers that recognize the Maf recognition element (MARE). CsMBE and MARE share a conserved core sequence but they differ in the 5′-adjacent nucleotide neighboring the core. Because of the high similarity between the CsMBE and MARE sequences, it has been unclear how many target binding sites and target genes are shared by the Nrf2-sMaf heterodimers and Maf homodimers. To address this issue, we introduced a substitution mutation of alanine to tyrosine at position 502 in Nrf2, which rendered the DNA-binding domain structure of Nrf2 similar to Maf, and generated knock-in mice expressing the Nrf2^A502Y mutant. Our chromatin immunoprecipitation-sequencing analyses showed that binding sites of Nrf2^A502Y-sMaf were dramatically changed from CsMBE to MARE in vivo. Intriguingly, however, one-quarter of the Nrf2^A502Y-sMaf binding sites also bound Nrf2-sMaf commonly and vice versa. RNA-sequencing analyses revealed that Nrf2^A502Y-sMaf failed to induce expression of major cytoprotective genes upon stress stimulation, which increased the sensitivity of Nrf2^A502Y mutant mice to acute acetaminophen toxicity. These results demonstrate that the unique cistrome defined as CsMBE is strictly required for the Nrf2-sMaf heterodimer function in cytoprotection and that the roles played by CsMBE differ sharply from those of MARE.